Since the early 1980s) there has been a increase in disease caused by organisms called nontuberculous mycobacteria (NTM), which is the generic name for mycobacteria other than M. tuberculosis and M. leprae (MOTT). They affect both immune-competent and immune-compromised persons, and patients with the human immunodeficiency virus (HIV) are known to be especially vulnerable. The most frequent NTMs involved in disease cases are known to be M. avium, M. intracellulare, M. scrofulaceum, M. kansasii, M. fortuitum complex, M. chelonae, M. abscessus, M. szulgai, M. malmoense, M. marinum, M. ulcerans, and M. africanum, M. bovis (28). Clinical diagnosis and treatment of nontuberculous mycobacterial infections are an increasingly frequent challenge to clinicians.
Currently, clinical diagnosis of mycobacteria to the species level is primarily based on cultural and biochemical tests. These conventional tests take several weeks, and the tests sometimes fail precise identification. The procedures for these tests are complex, laborious, and are usually impeded by the slow growth of mycobacteria in clinical laboratories. Additional methods, such as high-performance liquid chromatography, gas-liquid chromatography, thin-layer chromatography (5,21 36), and DNA sequencing analysis (3, 4, 15, 16, 17, 19, 26, 31, 32) can differentiate mycobacteria to the species level, but these are labor-intensive and difficult to perform for routine use in many clinical laboratories.
In contrast to the above-mentioned techniques, recent molecular techniques employing PCR-amplified products offers an easy, rapid, and inexpensive way to identify several mycobacterial species in a single experiment. PCR-restriction fragment length polymorphism analysis (PRA) has been developed to target mycobacterial genes, which are present in all mycobacteria such as hsp65 (7, 11, 25, 29, 30, 34, 35), 16S rRNA (2, 14, 37), and dnaJ (33). However, these techniques are still cumbersome since they require several enzyme digestions for species identification, and the results are not easy to interpret for species identification due to the limited size variation of DNA fragments after digestion.
On the other hand, probe-hybridization technique which employs DNA of the clinical specimen and oligo-probe hybridization (8, 9, 10, 18, 20, 23) is a useful tool for direct and rapid identification of NTM species. However, commercial kits currently available in the market are very expensive, limited only to 5 mycobacterial species, and the identification of a single species requires an independent experiment.